LOCUS       sequence-410539-        5290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SARS-CoV-2 Nsp3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5290)
  AUTHORS   Yang M, Mariano J, Su R, Smith CE, Das S, Gill C, Andresson T, 
            Loncarek J, Tsai YC, Weissman AM
  TITLE     SARS-CoV-2 papain-like protease plays multiple roles in regulating 
            cellular proteins in the endoplasmic reticulum.
  JOURNAL   J Biol Chem. 2023 Oct 12;299(12):105346. doi: 
            10.1016/j.jbc.2023.105346.
  PUBMED    37838170
REFERENCE   2  (bases 1 to 5290)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem."; date: "2023-10-12"; volume: "299(12):105346. doi"; pages: " 
            10.1016/j.jbc.2023.105346"
COMMENT     Sequence Label: pCMV-SARS-CoV-2-Nsp3PLpro
FEATURES             Location/Qualifiers
     source          1..5290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        65..256
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     213..233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(258..362)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      389..844
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(476..495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     686..707
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    851..972
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(862..881)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     916..935
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1246..1265
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1246..1264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1320..1336
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1321..1337
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2351..2374)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2374..2383
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2418..2436)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(2482..2685)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2511..2531)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2686..2989
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3164..3752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3244..3263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4081..4128
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4153..4172
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4360..5154)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4360..5154)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4471..4490)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5081..5100
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(5189..5290,1..256))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(5203..5290,1..48)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
       61 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
      181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagctg
      241 gttctttccg cctcaggatt cttccttttt caatattatt gaagcattta tcagggttat
      301 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
      361 cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg
      421 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
      481 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      541 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      601 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      661 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
      721 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
      781 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
      841 atttacgcgt taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
      901 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
      961 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     1021 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcat
     1081 gaacagactg tgaggactga ggggcctgaa atgagccttg ggactgtgaa tctaaaatac
     1141 acaaacaatt agaatcagta gtttaacaca ttatacactt aaaaattgga tctccattcg
     1201 ccattcaggc tgcgcaactg ttgggaaggg cgatcgagtg aattgtaata cgactcacta
     1261 tagggcgaat tgggtacact tacctggtac cttaattaat taaggtaccg ggccccccct
     1321 cgaggtcgac ggtatcgata agcttgatat cgaattcctc gagttacttg taggtcacgg
     1381 gcttgatggt ggtggtgtag ctgttctcct tgtagaacac gtcggtgatg ggacccttgt
     1441 attcagagga cttggtcagc agggctccgt cgatgcagta cagggtctcc ttagaggtga
     1501 tgtgcttgta gtgtccgcac tggtagttgc cggtgtactc ggaagcgcag gtgaaggtgc
     1561 cgtgcttcag ttcgtactgg gctggaggag cggacatcat cacgaatggg ctttcctgct
     1621 gcaccaggta cttggtggcc tgctttccgc aggtgcaagg gatctgcacg cccttcttga
     1681 actgctcgta ggacagggta cccatgtaca tcacagcttc cactcccttc agggtggtct
     1741 gctgctggcc gcaggtcttg cacaccacgt tcagcacacg cttgcaggag tccaggttgg
     1801 cgtgctggaa caggtagctc atggtctctc tcacgtctcc cagttcgccc acggtcttgt
     1861 tgcagtaggc caggatcaga gcgcagaagt tagcggcctc gccagcacgg gctctgtagt
     1921 aagcgtcctg cagagcgggt gggttgaact tcagttcgat ctgctgcagg gtcagcagag
     1981 cggtggccag gtagcagttg ttgtcggccc acttgatgct ggtcagtccg ttcacctggg
     2041 ggtacttcca cttcttggtg tggttcaggg cagacatgta cctgcccagg aaggatgggt
     2101 cggtggtgtg gtagtattcg aaagcctcca ctctcagggt gtcatcgtta ggcagcacgt
     2161 agaaggtctt gccctcgtgg gagttgtgtg gcttgatctt ggtcacgtca gctccgtcca
     2221 ggtaggtagg accgaactgc tgtccgtagg tcatgctcat gtccaccacc tgggtgtgca
     2281 ggttgatgtt gtccacggtg gtgaacacct tgatggtcct cacctcgcgc agagaggatc
     2341 cgcccgggct cttatcgtcg tcatccttgt aatccatggt ggcggccgcc accgcggtgg
     2401 agctccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcgctagcg
     2461 gatctgacgg ttcactaaac cagctctgct tatatagacc tcccaccgta cacgcctacc
     2521 gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg
     2581 gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc
     2641 aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat
     2701 gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata
     2761 atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata
     2821 cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg
     2881 gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg
     2941 gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat
     3001 atatgggcta tgaactaatg accccgtaat tgattactat taataactaa tgcatggcgg
     3061 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3121 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3181 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3241 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3301 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3361 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3421 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3481 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3541 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3601 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3661 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3721 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3781 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3841 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3901 atgagtaacc tgaggctatg gcagggcctg ccgccccgac gttggctgcg agccctgggc
     3961 cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg tattggcccc
     4021 aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat
     4081 gaacaaacga cccaacaccg tgcgttttat tctgtctttt tattgccgtc atagcgcggg
     4141 ttccttccgg tattgtctcc ttccgtgttt cagttagcct ccccctaggg tgggcgaaga
     4201 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
     4261 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
     4321 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     4381 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     4441 gtcagcccat tcgccgccaa gttcttcagc aatatcacgg gtagccaacg ctatgtcctg
     4501 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     4561 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     4621 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gttcttcgtc
     4681 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     4741 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     4801 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     4861 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     4921 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
     4981 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     5041 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     5101 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     5161 cgatcctcat cctgtctctt gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc
     5221 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa
     5281 aaattagtca
//
